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Lentiviral Vectors
Virus Characteristics : Lentiviruses are medium-
sized (120 nm), enveloped viruses composed of a
nucleocapsid containing two copies of single-stranded
positive-sense RNA.
LENTIVIRUS
Lentivirus is a genus of slow viruses (lente-, Latin for
"slow") of the Retroviridae family, characterized by a
long incubation period.
The viruses are species-specific in host range and
several have been recognized as pathogens of domestic
animals, non-human primates and humans.
Although viral vector systems based on Feline
Immunodeficiency Virus (FIV) and Equine
Infectious Anemia Virus (EIAV) are available,
lentiviral vectors are typically based on the best
characterized of all lentiviruses: Human
Immunodeficiency Virus serotype 1 (HIV-1).
HIV sculpture - Luke Jerram
Lentiviruses can deliver a significant amount of
genetic information into the DNA of the host cell
and have the unique ability among retroviruses of
integrating into the genome of non-dividing cells.
The viral genome remains in the host genome
and is passed on to the progeny of the cell when
it divides. For this reason, they are one of the
most efficient methods of a gene delivery vector.
Advantage of lentiviruses over
gamma-retroviruses: They do not
require mitosis for productive infection.
Last update: Feb 2014
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Genome - HIV
The genomes of lentiviruses are complex, encoding a number of regulatory and accessory proteins not
found in other retroviruses. The information provided in this section will be focused on the HIV-1.
In addition to the gag, pol and env genes common to all retroviruses, HIV-1 contains two regulatory
genes, tat and rev, essential for virus replication and four accessory genes, vif, vpr, vpu and nef that,
while dispensable for virus growth in vitro, they are critical for in vivo replication and pathogenesis.
The ~10 kb HIV genome is flanked by long terminal repeats (LTRs) which are noncoding
sequences that play an important role in virus replication and gene transcription. LTRs
include R, U5 and U3 sequences.
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Adapted from The Immune System, 3rd ed. (© Garland Science 2009)
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HIV Routes of Transmission
HIV is primarily spread through direct contact of the virus
with mucous membranes and broken skin (e.g. scratches,
cuts, abrasions, dermatitis, or other lesions). Percutaneous
(e.g. needle stick) exposure is also an important route of
transmission.
The clinical manifestations of HIV infection is
divided into three different stages: initial, chronic
and final.
The symptoms of the initial stage are very
unspecific. Individuals usually present with flu-
like syndrome after two weeks of infection
followed by the chronic stage which no
symptoms are present. The final stage of HIV
infection corresponds to AIDS. AIDS symptoms
will vary according to the acquired opportunistic
infection and/or malignancy developed.
Infection occurring via the respiratory tract has not been
documented, and is unknown. However, since many of
lentiviral vectors are pseudotyped with the glycoprotein of
the Vesicular Stomatitis Virus, which can be transmitted by
aerosol route, this route should be considered as well.
Clinical Manifestations - HIV
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Cell Tropism - HIV :
HIV can infect a variety of human immune
cells such as CD4+ T cells,
macrophages, and microglial cells.
Viral entry to target cells is mediated
through interaction of the virion envelope
glycoproteins (gp120) with the CD4
molecule and chemokine co-receptors of
the host cells (CCR5 or CXCR4).
Altering Cell Tropism - Pseudotyping
CD4 is the major receptor for the native HIV envelope glycoprotein and, therefore, the tropism for lentiviral
vectors with this envelope protein is highly restricted. In order to infect cells without CD4 expression,
several heterologous envelope proteins have been used for pseudotyping.
The Vesicular Stomatitis Virus glycoprotein G
(VSV-G) is preferentially used to allow gene
transfer to a broad range of cell types and species
(from insects to humans). A further advantage of
VSV-G pseudotyping is the increased stability of
the viral particles, which enables concentration of
the viral particles by ultracentrifugation.
Other envelopes used for pseudotyping lentiviral
vectors may include envelope proteins of other
retroviruses, such as human foamy virus, gibbon
ape leukemia virus (GALV) and the feline
endogenous virus (RD114). All still maintain the
tropism of the vector for human cells.
For transfection of murine cells, lentiviral vectors
can be pseudotyped with a murine ecotropic
envelope . This would remove exposure risks to
the workers, since they cannot infect human cells.
Although pseudotyping a lentiviral vector with VSV-G provides advantages, this increases the risks of infection following an accidental exposure where not only CD4 + cells are going to be the target, but any cells in the site of inoculation.
EM of VSV particles
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Although viral genomic integration is essential to obtain stable expression of the gene of interest, it may potentially contribute to insertional mutagenesis.
The integration of the provirus can
disturb the function of cellular genes
and lead to activation of oncogenes or
inactivation of tumor suppressors
promoting the development of cancer.
Lentiviruses are unique among the members of the family in the fact that they can infect non-dividing cells (e.g. cells from the nervous system, muscle and liver cells) by actively entering the nucleus of a cell through the nuclear pore. This feature greatly expands the scope of potential gene transfer applications.
Lentiviral Genome Integration : A double edged sword
Members of the Retroviridae family, including
lentiviruses, have the ability to integrate into the host
chromosome during their replication cycle.
Even a non-replicative lentiviral vector, carrying a “non-harmful” gene
(e.g. GFP) or gene inhibitor, in theory, can cause harm.
Provirus : name given to the viral nucleic acid when integrated into the host chromosome,.
As cell divides, the provirus is transferred through generations.
Lentiviral vectors can be produced through the use of mutations in the integrase protein that minimize proviral integration. The resulting integrase-deficient lentivirus (IDLV) generates circular vector episomes in transduced target cells that are gradually lost by dilution in dividing cells (transient expression), but are stable in quiescent cells. Inherently, IDLVs have a greatly reduced risk of causing insertional mutagenesis compared to integrating lentiviruses.
Integrase-Deficient Lentivirus: Circumventing oncogenic potential
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New viral RNA and proteins move to cell surface and a new, immature, HIV virus forms and leaves the cell through “budding”.
The risk of insertional mutagenesis due to
chromosomal integration is a safety concern. The
provirus can disturb the function of cellular genes and
lead to activation of oncogenes or inactivation of tumor
suppressors promoting the development of cancer.
Fusion of the HIV to the host cell surface.
Viral RNA is converted
to DNA by the Reverse
Transcriptase (RT)
enzyme.
Viral DNA is transported across the nucleus and integrates into the host DNA (provirus).
Transcription of the provirus DNA into RNA to generate genomic RNA and to make viral proteins.
Viral particles mature by the protease
enzyme which modifies viral protein
chains to make the particle ready to
infect other cells.
Lentiviruses are
unique among the
Retroviridae family in
the fact that they can
infect non-dividing
cells by actively
entering the nucleus
of a cell through the
nuclear pore. Even a non-replicative lentivirus, carrying a “non-harmful” gene (e.g. GFP) or
gene inhibitor, in theory, can cause harm.
HIV – Viral Cycle Steps
Virus enters the cell, capsid is “broken” and
RNA, enzymes and other viral proteins are released
into the cytoplasm.
HIV particles budding from
infected cell
6
5
7
This is shown only if the Replication Cycle link is clicked
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Lentiviruses are known as having high
mutation and recombination rates and the
possibility that the HIV could self-replicate
and could be produced during vector
manufacture by recombination process is a
real concern.
To reduce the reversion probability, viral accessory genes (i.e. vif, vpr, vpu and nef) are deleted and
essential genes have been separated in different plasmids, so that multiple recombination events would
be required in order to form a replicative competent lentivirus (RCL).
Lentiviral Vector Production
Generation of recombinant
lentiviral vector particles
involves subcloning the
gene of interest (transgene)
into an HIV-1 Transfer Vector
backbone, which is co-
transfected with Helper
Plasmids into a recipient cell
line (Packaging Cell) .
Generation of recombinant HIV-1 vector particles
involves subcloning the gene of interest into an
HIV-1 transfer vector backbone, cotransfection of
the plasmid into a recipient cell line together with
packaging and envelope plasmids (or directly into
a stable packaging line), and collection of vector
supernatants.
Helper Plasmids
Helper Plasmids
Vector Packaging:
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Transfer Vector (Viral Construct) The genetic information contained in the Transfer
Vector genome is the only one transferred to the
target cells. In this construct, the gene of interest (or
gene inhibitor) is cloned into a vector sequence that
contains the Packaging Signal (Psi-sequence - Ψ)
and is flanked by the viral LTRs.
The LTRs are necessary since they are the control center for gene expression. Packaging signal (Ψ) is
the element required for encapsidation of the viral genome, in other words, it indicates in which genome
structural proteins should be assembled.
Helper Plasmids
Genes for structural viral proteins (gag) and
enzymes (pol), as well as regulatory genes (tat and
rev), are provided in plasmid(s) separated from the
one with the transgene or gene inhibitor (Transfer
Vector). The number of the Packaging Plasmids
varies according to the vector system.
Packaging Plasmid(s)
Envelope Plasmid In lentiviral packaging systems, the native envelope (HIV env
gene) is typically replaced with a helper plasmid expressing
heterologous envelope glycoproteins (e.g. VSV-G).
Packaging Cell - The vector “factory”
This is the location where the viral vector
production will take place. To produce a lentiviral
vector, several plasmids are transiently transfected
into a packaging cell line, commonly HEK293 cells.
The final viral vector particle will not contain any of the HIV genes provided by the helper plasmids,
only their proteins. Without the essential genes, the viral vector will not be able to replicate, but still has
the ability to infect cells and to have its transgene expressed. Depending on the nature of the
transgene, a vector may be harmful following an accidental exposure.
HEK 293 cells are transformed cell
lines derived from human embryonic
kidney preparations.
Lentiviral Vector Production : Packaging
Helper plasmids lack the packaging signal, therefore, their genes will not be enclosed by the viral capsid.
Although vector is rendered replication-incompetent, it remains infective !
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rev
Lentiviral vector production systems have been
refined over time to improve their performance and
safety. Depending on their features, vector systems
are divided into different generations.
First-generation - The first-generation lentiviral vectors were manufactured using a packaging
system that comprised all HIV genes, except the env gene (usually heterologous), which is separated in
one plasmid.
gag, pol, tat, rev and accessory genes
Second-generation - It was shown subsequently that none of the four HIV-1 accessory genes
vif, vpr, vpu, or nef were required for HIV-1 replication in immortalized cell lines. This led to the
development of a “second generation” of HIV-1 vector systems.
The higher the generation, the safer the vector.
In this system, the accessory genes were eliminated leaving the gag and pol reading frames,
which encode for the structural and enzymatic components of the virion, respectively, and the
tat and rev genes, fulfilling transcriptional and post-transcriptional functions.
env (het.)
gag, pol, tat, rev In general, lentiviral vectors with
a wild type 5’ LTR need the 2nd
generation packaging system
because these vectors require
tat for activation.
Third-generation / Self–Inactivating (SIN) In a third-generation system, only gag, pol, and rev
genes remain present (tat is eliminated). The rev gene is provided in a separated plasmid. Since the
HIV promoter in 5’ LTR depends on tat, a vector which lacks tat needs to have its wild type promoter
replaced with a heterologous enhancer/promoter such as CMV or RSV to ensure transcription.
Lentiviral Vector Generations:
gag, pol
rev
Transfer Vector
transgene*
transgene*
Transfer Vector
Transfer Vector
transgene*
* The Transfer Vector may also carry oligonucleotides (e.g. micro RNA, shRNA) that will inhibit target gene(s) of the host.
Packaging Plasmid
Packaging Plasmids
Packaging Plasmid Envelope Plasmid
Envelope Plasmid
Envelope Plasmid
env (het.)
env (het.)
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First
generation
Second
generation
SIN Third generation
Number of Plasmids 3 3 4
Deletion in the 3'LTR - SIN
("self-inactivation") NO NO YES
Number of Packaging
Plasmids containing HIV genes
1 1 2
Accessory genes
vif, vpr, vpu, nef
All present All absent All absent
tat and rev genes
tat and rev are
present on a single
packaging plasmid
tat and rev are
present on a single
packaging plasmid
tat is absent, rev
protein is expressed
from a separate
plasmid
gag and pol genes
On the same
plasmid
On the same
plasmid
On the same
plasmid
Recombination events to
generate Recombinant
Competent Lentiviruses (RCL)
2 recombinations 3 recombinations
4 recombinations,
between plasmids
without homology
and pick up of a
promoter to
complement
'SIN' deletion
Lentiviral Vector Generations : Summary Table
Table adapted from Pauwels et al - Current GeneTherapy,2009,Vol.9,No.6.459-474
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The Viral RNA LTR contains two
parts: the unique sequences (U5 at the
5'- end and U3 at the 3'- end); and the
repetitive sequence (R - located at
both ends of the RNA genome).
Provirus (DNA):
When viral RNA is
reverse transcribed to
DNA, a U3 region is
added to the 5'-LTR, and
a U5 is added to the
3'-LTR, resulting in two
identical terminal
structures.
Transcript (RNA):
Transcription of the
provirus generates a
RNA with the same
LTR structure as the
original Viral RNA.
SIN vectors have modifications in their 3’ Long terminal repeat (LTR).
Self-Inactivating (SIN) Vectors: Increasing Safety
What are LTRs?
LTRs are noncoding sequences that are located on each end of the HIV genome.
The U3 region is in fact the functional HIV-1 promoter, and contains the transcriptional enhancers.
The R region marks the starting point of transcription, while the U5 region is involved in reverse
transcription.
Although the recombination event possible between three independent plasmids is extremely small,
ways to decrease this probability even further when dealing with HIV-1 based vectors have been
undertaken. This is the basis of the development of self-inactivating vectors or SIN vectors.
In a SIN vector, the promoter sequences in the U3 region of the 3’LTR is deleted (Δ3'-LTR). When
a vector infects a target cell, during the process of reverse transcription (RNA to DNA), the 3’LTR
is copied to the 5’LTR and the 5'-LTR of the provirus will lack an active viral promoter.
By design, SIN vectors require an internal heterologous promoter for transgene expression. This
can be either a viral (e.g. CMV, Rous sarcoma virus) or a cellular promoter (e.g. EF1-α ).
Adapted from Future Microbiol © 2010 Future Medicine Ltd
This is shown only if the LTR link is clicked
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Advantages
Stable integration into the host genome
with stable expression of the transgene
Ability to carry transgenes of reasonable
sizes (up to 8Kb)
Infect dividing and non-dividing cells
(different from gamma-retroviruses)
Efficient gene transfer
No immunogenic proteins generated
Lentiviral Vector & Human Derived Materials
The wild type HIV may recombine with the vector
potentially leading the creation of novel viruses with
unpredicted potential for diffusion and pathogenesis.
Experiments involving human materials unscreened for HIV
must be performed with extreme precaution.
The risk of RCLs formation exists not only during viral
vector production, but also during experiments involving
materials infected with wild type HIV.
Lentiviral Vector Application
Disadvantages
Potential for generation of replication
competent lentivirus (RCL)
Oncogenesis potential
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Due to the presence of a lipid envelope in their structure, lentiviral vectors are rapidly inactivated when
exposed to drying environmental conditions. They are susceptible to most of common use disinfectants,
such as 70% ethanol.
Artwork of HIV particle budding from
infected cell and carrying its cytoplasmic
membrane (viral envelope).
Alcohol based disinfectants are not
recommended, however, for spill clean-
up due to evaporation, which results in
brief contact times. Also, they have a
limited activity in the presence of organic
material.
A mixture of 70% ethanol or isopropanol
diluted in water is effective against a
wide spectrum of organisms, including
enveloped viruses.
Lentiviral Vector: Environmental Stability
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Containment
Although the parent virus for most of lentiviral
vectors belongs to the Risk Group Classification 3
(i.e HIV), in general, guidelines recommend
BSL2 practices for working with these agents.
The implementation of BSL2 measures is
adequate for the production or handling of most
replication-defective lentiviral vectors, unless
large volumes are exceeded (according to NIH
this is >10L production volumes).
The handling of lentiviral vector suspensions,
in particular high titer stocks and/or volume,
increases the possible accidental exposure of
the lab worker and therefore is considered to be
an activity associated with higher risks. In such
case, BSL2 facilities with BSL3 practices should
be considered (i.e. BSL2+).
Handling first generation lentiviral vectors may also
require the implementation of additional measures
because these vectors are considered to be less safe
and the probability of RCL is not negligible.
BSL2 lab
Non-human lentiviruses such as FIV and EIAV are
classified as Risk Group 1 organisms. Although BSL1
facilities and practices are normally appropriate for this
Risk Group class, it is important to note that those vectors usually are pseudotyped with a human tropic envelope, such as
the VSV-G envelope . In this case, BSL2 containment must be
implemented since these viruses now have the capability of
transducing human cells.
Lentiviral Vector: Laboratory Activities
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Lentiviral Vector: Laboratory Activities
Containment: Animal Experiments
Animals that do not support replication of
HIV-1 that are injected with safer lentiviral
vectors systems (e.g. SIN third generation
vectors), can be housed in an ABSL1 lab
after a period of time that ranges between 1
to 7 days.
The recombinant DNA advisory Committee
(RAC) of the National Institutes of Health
(NIH) recommends ABSL2 containment for
animals that are not permissive for lentiviral
infection (e.g. mice, rats).
At the University of Cincinnati, animals receiving
lentiviral vectors must be housed in ABSL2 conditions
for a period of 72 hours.
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Lentiviral Vector: Laboratory Activities
Exposure Risks
Percutaneous Exposure - The major potential
hazard is infection of the researcher by parenteral
inoculation (e.g. needle stick accidents). The use of
sharps including needles, blades and glassware should
be strictly limited. Strongly consider the use of
engineered sharps systems. If feasible, use plastic
disposable transfer pipettes rather than glass Pasteur
pipettes.
For activities conducted outside of a
biosafety cabinet (e.g. stereotactic injection),
the use of mucous membrane protection
devices is of extreme importance .
Proper restraint technique (physical or chemical)
is critical to minimize accidental exposure during
in vivo experiments involving sharps.
Open wounds, cuts, scratches, and grazes should be
covered with waterproof dressings in addition to PPE.
Mucosal Exposure - Laboratory personnel may
also be exposed by direct contact of vector suspension
(e.g. splash) with oral, ocular or nasal mucosa. Extreme
care must be taken to avoid spilling and/or splashing
infected materials, especially if working with high
volumes (>10L). Lentiviral vector manipulations should be
conducted in a biological safety cabinet
which not only provides respiratory protection,
but also protects the worker against splash.
The use of face shields provides protection against ocular, nasal
and oral mucosa exposure. If eye protection is used (e.g.
goggles), nasal and ocular protection can be achieved by the use
of surgical masks.
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Post Exposure Prophylaxis : PEP
The current guidelines for managing
occupational exposure to HIV-based viral
vectors rely on the use of antivirals. In this
type of exposure, antivirals should target the
pre-integration steps of the viral cycle to
prevent insertional risks.
Ideally, it is recommended that post-exposure
prophylaxis (PEP) be administered
immediately, but certainly no later than 72h
following exposure .
Antivirals that inhibit the viral Reverse
Transcriptase (RT) are used as PEP drugs. When
RT is blocked, DNA copy cannot be formed from the
viral RNA. Also, inhibitors of the viral Integrase may
be added to the regimen (see replication cycle).
Reverse Transcriptase (RT) converts the HIV RNA
genome into DNA for integration into a host cell genome.
Each PI should develop an emergency response plan in case of exposure to lentiviral vectors. It is the
responsibility of the PI to provide risk assessment information if emergency department providers have
any questions regarding health hazards.
PEP treatment needs to be prescribed by an
occupational health physician who must determine
whether treatment is necessary based on the biological
hazards associated with the expressed transgene.
Following an accidental exposure, a lentiviral vector can potentially infect the lab worker cells. This could
not only result in permanent transgene expression with its associated harmful effects, but might also
result in activation (or inhibition) of neighbouring
genes due to insertional mutagenesis.